Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths in the world, accounting for over 1 million cases annually. In the United States its incidence has increased from 1.4 (1976-80) to 2.4 (1991-95) per 100,000, which may be related to an increase in chronic hepatitis C infection, with an estimated 16,600 new cases and 14,100 deaths in 2002. Median survival of untreated HCC patients is only 7.8 months, with a 3-year survival rate of 10%. The current treatment modalities for HCC are hepatic resection, intra-tumoral ablation and orthotopic liver transplantation, which are not very effective and patient's prognosis remains poor. Conditionally replicating viruses targeted to tumors are being developed as a novel class of oncolytic agents. Vesicular Stomatitis Virus (VSV) is a negative-strand RNA virus with inherent specificity for replication in tumor cells due to their attenuated anti-viral responses. VSV as an oncolytic virus is particularly appealing for its exceptionally rapid replication rate of 1-2 hours in tumor cells, such that the oncolytic effects could be maximally manifested before the onset of potentially neutralizing anti-viral immune responses in the host. We have successfully rescued a recombinant VSV vector expressing the green fluorescent protein (rVSV-GFP) and demonstrated its cytopathic effects and replication in cultured Morris rat hepatoma cells and in solitary nodules of HCC established in the livers of syngeneic Buffalo rats. We propose to investigate the molecular mechanism(s) by which VSV's inherent tumor specificity is manifested in the rat hepatoma cells. In order to assess the tumoricidal potential of VSV to treat multi-focal HCC in an immune-competent host, such a model has been developed in the livers of syngeneic Buffalo rats through mesenteric vein infusion of the Morris rat hepatoma cells. Three days after hepatic artery infusion of rVSV-GFP, extensive necrosis was apparent in multiple hepatic lesions that were also positive for GFP by flouresence analyses and VSV by immuno-histochemical staining. Importantly, the surrounding normal liver tissues were negative for VSV staining and any signs of pathology. Using this immune-competent syngeneic animal model with multi-focal HCC in the liver, we propose to test the following hypotheses: 1. rVSV, delivered through hepatic artery infusion, can be an effective oncolytic agent and prolong survival of the tumor-bearing animals with minimal toxicities; 2. Its oncolytic potential can be enhanced by incorporation of the HSV-VP22/TK fusion gene into the vector with timely administration of ganciclovir; 3. Its effectiveness can be further amplified by incorporation of a heterologous viral fusogenic membrane glycoprotein gene that will induce syncytia formation with neighboring cells and 4. Repeated administration of the oncolytic virus can be effectively applied in immune-competent animals by substituting its G gene for those from VSVs of different serotypes. Successful conduct of these pre-clinical studies will provide the scientific foundation for future development of recombinant VSV as a novel therapeutic agent for multi-focal HCC in patients.